Blocking a gram-negative bacteria from expressing a virulence factor

ABSTRACT

Methods of and compositions for producing and using plant-based materials are provided. The methods include using biopolymers or their synthetic equivalents combined with a stable source of reactive oxygen species that when applied to or combined with a separate source of oxido-reducing enzyme or catalyst will cause the formation of an activated biopolymer with increased protein binding affinity and microbial control activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/878,239, filed Jan. 23, 2018, which is a continuation of U.S.application Ser. No. 14/178,459, filed Feb. 12, 2014, which is acontinuation of U.S. application Ser. No. 14/027,228, filed Sep. 15,2013, which is a continuation of U.S. application Ser. No. 13/680,007,filed Nov. 16, 2012, now U.S. Pat. No. 8,586,110, which is acontinuation of U.S. application Ser. No. 12/715,270, filed Mar. 1,2010, now U.S. Pat. No. 8,343,552, which claims benefit of U.S.Provisional Application No. 61/209,260, filed Mar. 4, 2009, and is acontinuation-in-part of U.S. application Ser. No. 12/317,638, filed Dec.23, 2008, which claims benefit of U.S. Provisional Application No.61/009,484, filed Dec. 28, 2007; each application of which is herebyincorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention generally relates to controlled enhancement ofprotein binding affinity biomaterials. More in particular, the inventionrelates to stabilization of and controlled activation of plantbiopolymers by enzymes of animals, plants, bacteria, and/or othercatalysts to cause locally enhanced oxidation and/or cross-linking ofproteins, micro-organisms and biologic tissues.

BACKGROUND OF THE INVENTION

Despite hundreds of millions of years of divergent evolution, almost allplants, animals and pathogens share some common biochemical fundamentalsand strategies for environmental defense. This makes botany a richsource of useful and compatible compounds for the control of pathogensin animals. The use of functional biochemistry from the plants has longbeen the basis for traditional and herbal medicines and often consideredless likely to trigger unwanted immunological responses between lessgenetically distant species within the same phyla than highly purifiedlarge complicated proteins or polymers.

Immune systems of most of the higher organisms protect from infectionwith defenses of increasing specificity. The simplest is a physicalbarrier that prevents pathogens, such as bacteria and viruses, fromentering the organism. Plants and animals also have innate immunesystems that are either genetically coded responses to specificpathogens, or various non-specific responses to pathogen chemistries.

Plants typically have a two branched immune system. The first recognizesand responds to molecules common to many classes of microbes, includingnon-pathogens, by increased expression of ROS (Reactive Oxygen Species)generating enzymes capable of initiating oxidative bursts, but suchdirect oxidative response is energy costly and must be strictlyregulated to prevent autotoxicity. Many pathogenic microorganisms(bacteria, fungi, protozoa) are equipped with peroxidases or catalasesas countermeasures against such ROS bursts. The second branch of theinnate immune system is the multi-component wound response as describedabove initiated by the reaction between quinone compounds and aminoacids when cells are damaged. These compounds are usuallycompartmentally separated and do not cooperate in living systems. Inplants, cellular disruption causes various phenol compounds and reactiveoxygen species to come into contact with polyphenol oxidases (PPO),oxidizing the phenol compounds to firm quinonic compounds thataggressively associate with each other and amino acids of the cells orany microorganisms present. This effects many physiologic phenomena,such as browning or discoloring of foods, precipitation of proteins,germicidal activity, astringency, changes in food digestibility andmore.

Polyphenol oxidation in plant systems generates oxidized-polyphenols(also referred to as o-polyphenols, oxidized biopolymers, polyquinonesand quinonic compounds) with a multiplicity of quinonic groups that arecapable of covalent bonding. Once formed, the high affinityo-polyphenols spontaneously form covalent intra- and inter-chaincross-links that condense proteins far more aggressively than hydrogenbonds characteristic of non-oxidized polyphenols. In plant systems,o-polyphenols cross link damaged cell proteins to form a refractoryshield between the healthy tissues and further assault. They alsoprevent pathogen propagation by aggressively binding to their metabolicpathways, disabling virulence enzymes and arresting pathogen motility.

Higher vertebrates possess an additional layer of protection, theadaptive immune system, which allows for a stronger immediate immuneresponse to previously encountered pathogens. The aggregation of smallermolecules on the pathogen creates large complexes with an increasedantigenicity of the pathogen to the host immune system. Each pathogen is“remembered” by a signature antigen. Should a pathogen infect the bodymore than once, these specific memory cells are used to quickly andefficiently eliminate it; however, these tailored responses can takemany days to develop. In the interim, primary defense against newlyencountered pathogens, especially in infection of immunologicallydeficient or immature animals relies solely on the innate immune systemsand often is associated with negative physiologic responses such asdiarrhea, vomiting, fever, inflammation, etc. Such systemic responses toinfection are the expression of the very large numbers of immuneeffectors that can be extremely metabolically expensive, even fatal tothe host.

One of the most common dangers associated with an unchecked systemicresponse by the innate immune system is diarrheal dehydration triggeredby infectious diseases or parasites. Diarrheal dehydration affects over2 billion people each year and is the most common cause of death forThird World infants, responsible for over 1.5 million deaths per year.Besides re-hydration, most efforts to treat diarrhea have focused onincreasing human mucosal immunity by modulating systemic immuneresponses, such as by using intestinal motility reducing drugs, mucouspermeability modifiers or antibiotic therapies. These approaches havelimited success but introduce undesirable risks of side effects,pathogen resistance, or physiologic senescence.

There is constant commercial demand for botanical alternatives toantibiotics and synthetic chemical disinfectants for the control ofdisease associated with water, surface, and food borne pathogens. Theexplosive rise in antibiotic resistant diseases has been associated withthe overuse of antibiotics in both humans and livestock. Many regionalgovernments and international health organizations have called for phaseout of unnecessary antibiotic use, especially in livestock feeds wherethey are used sub-therapeutically to enhance growth. To date, it iswidely recognized that there are few cost effective and environmentallysound alternatives for the safe control of pathogens. Decades ofresearch on plants as sources of new antimicrobials has primarilyfocused on mechanical or solvent extraction of specific plant compoundsand has not been successful in generating compositions with potency,safety, user preference and environmental profile necessary to match theperformance of current antibiotics and germicides.

SUMMARY OF THE INVENTION

In an aspect, a biochemical composition comprises a processed fluidcontaining a molecule having a hydroxyl group is combined with anactivating mechanism to activate the molecule by oxidizing the hydroxylgroup with an oxidizing agent and a catalyst. Activating the moleculeincreases the binding affinity of the molecule.

In one embodiment, the molecule comprises a polyphenol. In analternative embodiment, the molecule comprises a polymeric carbohydratemolecule or polysaccharide derivatives. In another embodiment, thepolyphenol is derived from a plant, in an embodiment, the plantcomprises camellia sinensis, or punica granatum or other polyphenolbearing plants. In another embodiment, the polyphenol is derived fromthe root, leaves, stems, bark, fruit or other tissues of polyphenolbearing plants.

In an alternative embodiment, the molecule comprises tannin, lignin,flavonoid, hydroxycoumarin, or alkaloids. In another embodiment, themolecule comprises at least an artificial synthetic section. In anembodiment, the catalyst comprises a catalase, a peroxidase, aphenoloxidases, a tyrosinase, or a metal catalyst. In an alternativeembodiment, the catalyst is located at an animal cell. In anotherembodiment, the catalyst is generated by a pathogen. In an embodiment,the pathogen comprises virus, bacteria, fungi, an eukaryotic organism,or prionic. In an alternative embodiment, the oxidizing agent comprisesreactive oxygen species (ROS).

In another embodiment, the reactive oxygen species comprises hydrogenperoxide. In another embodiment the reactive oxygen species comprisesinorganic or organic peroxides. In an embodiment, the reactive oxygenspecies comprises a product of ozone reduction by superoxide dismutase,glucose oxidase, hydration of a percarbonate, or hydration of carbamideperoxide (urea peroxide) or other indirect method of generating stablereactive oxygen species. In an alternative embodiment, the processedfluid is prepared from a dry mixture containing a polyphenol or apolysaccharide derivative. In another embodiment, the processed fluid isprepared from intact plant material containing a polyphenol. In anotherembodiment, the activating mechanism is initiated when the polyphenol orthe polysaccharide derivative is in contact with the enzyme and theoxidizing agent in a solution. In an embodiment, the hydroxyl groupbecomes a carbonyl group after the activation of the hydroxyl group. Inan alternative embodiment, the molecule comprises a quinonic group afterthe activation of the hydroxyl group. In another embodiment, themolecule has an effect in inactivating a pathogen after the activationof the hydroxyl group. In an embodiment, the oxidizing agent providesfree radicals. In alternative embodiments, the activated moleculeprovides free radicals.

In a second aspect, a method of preparing a composition comprisesobtaining a biopolymer from a plant and activating a hydroxyl group onthe biopolymer, so that a molecular binding affinity of the biopolymeris increased.

In an embodiment, the biopolymer comprises a polyphenol, apolysaccharide derivative, or a polymeric molecule. In alternativeembodiments, the activating is performed by placing the biopolymer incontact with an enzyme or an oxidizing agent. In another embodiment, themethod further comprises cross-linking the activated biopolymer with aprotein of an animal. In an embodiment, the method further comprises aplurality of biopolymers forming cross-linking structures amongthemselves. In an alternative embodiment, the method further comprisesbinding the biopolymer with a pathogen. In an alternative embodiment,the method comprises binding the biopolymer with a cell. In anotherembodiment, the method comprises binding the biopolymer with a virus. Ina further embodiment, the binding of the polymer to multiple pathogenscauses agglomeration or removal of microorganisms, proteins orproteinaceous structures from solution. In another embodiment, thebinding ceases the propagation of the pathogen. In some embodiments, theactivated biopolymer is capable of blocking a metabolic pathway of apathogen. In an alternative embodiment, the method further comprisestreating a diarrhea symptom of an animal using the activated biopolymer.In a further embodiment, the method comprises treating any damagedtissue of an animal using the activated biopolymer. In anotherembodiment, the activating the hydroxyl group is triggered by an enzymeon a site of an animal. In an embodiment, the activating the hydroxylgroup is achieved by exogenous addition of an enzyme. In an alternativeembodiment, the method further comprises removing or inactivating anoxidoreductase or a reducing compound. that reacts with an oxidizer. Inanother embodiment, the method further comprises forming a barrier bycross-linking the biopolymer with a protein of an animal, so an invasionof a pathogen is prevented. In a further embodiment, the methodcomprises cross-linking the biopolymer with a protein of an animal, soas to promote accelerated wound healing.

In a third aspect, a method of facilitating a localized reactioncomprises localizing an added reactive oxygen species in a reactiveproximity of a hydroxyl group on a biopolymer, activating the hydroxylgroup of the biopolymer, and applying the biopolymer to a target site.

In an embodiment, the biopolymer comprises a polyphenol or apolysaccharide derivative. In an alternative embodiment, the activatingis achieved by causing an encountering of an enzyme. In anotherembodiment, the reactive oxygen species comprises hydrogen peroxide. Inan embodiment, the method further comprises increasing the density ofthe hydrogen peroxide in the reactive proximity of a hydroxyl group byadding hydrogen peroxide to a solution containing the biopolymer. In analternative embodiment, the biopolymer comprises multiple hydroxylgroups.

In a fourth aspect, a controlled chemical delivery method comprisespreparing a bio-molecule containing a pre-selected chemical substanceand selecting a size, a weight, or a combination thereof to control apenetration rate of the bio-molecule through an animal tissue.

In an embodiment, the chemical substance comprises a phenolic compound.In an alternative embodiment, the chemical substance comprises quinoniccompound. In another embodiment, the bio-molecule comprises some amountof an extract from a plant. In an embodiment, preparing the moleculecomprises isolating the molecule. In an alternative embodiment,selecting comprises extracting. In another embodiment, the penetrationrate is moderate, so that an application of the bio-molecule to ananimal does not cause a toxic response from the tissue of the animal. Ina further embodiment, an application of the bio-molecule to an animalreduces the external stimulation of immune response from the animalsystem. In an embodiment, the ingestion of the bio-molecule improves theutilization of nutrition for growth in animal systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several reaction pathways for phenol unit conversioninto quinones in accordance with an embodiment of the presentapplication.

FIG. 2 illustrates a composition prepared in accordance with oneembodiment of the present application.

FIG. 3 illustrates a flowchart of a method of preparing a plant-basedcomposition in accordance with one embodiment of the presentapplication.

FIG. 4 illustrates a flowchart of a method of inactivating reducingagents/enzymes using a solvent in accordance with one embodiment of thepresent application.

FIG. 5 illustrates a flowchart of a method heat inactivating reducingagents/enzymes in accordance with one embodiment of the presentapplication.

FIG. 6 illustrates applications of the plant-based composition inaccordance with some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises novel bioactive compositions. In anaspect, a biochemical system comprises a biopolymer or the syntheticequivalent combined with a stable source of reactive oxygen species(ROS) and a separate source of an oxido-reducing, enzyme or catalyst.The combinations of the substances are able to cause the formation of anoxidized biopolymer with increased protein binding affinity andmicrobial control activity.

Some embodiments of present invention contain a mixture of plant basedmaterial with astringent and/or germicidal properties and an activationprecursor in stable solution. The solution is substantially free ofenzymes and catalytic substances that can cause the other components toreact in a manner that causes degradation. The mixture of this plantbased germicidal material and activation precursor is catalyzed. byvarious enzymes of animal, plant or microbial cells to release oxidativeradicals and to form an activated plant based material withsignificantly enhanced astringent and germicidal properties. The releaseof oxidative radicals and the formation of activated plant material aregenerally localized to the catalyzing biologic enzyme source, thusconcentrating such activity in the proximity of the triggering biologicentity or material for maximum effect. The triggering catalyst can alsobe of non-biologic origins such as a metal that causes reduction of theactivation precursor to cause auto-oxidation of the plant based materialinto its activated form.

In alternative embodiments, the bioactive and germicidal system containsa mixture of a plant based material, such as polyphenols. A person ofordinary skill in the art would appreciate that any macromolecules,polymers, aggregate of small molecules, cellular membrane fragments,cross linked compounds containing a multiplicity of exposed phenolicunits and an activation precursor (e.g., an oxidizer, such as hydrogenperoxide), or combinations thereof are applicable.

In other embodiments, activator materials contain ozone or peroxone. Insome embodiments, the plant material can be a source of naturallyoccurring hydrogen peroxide. However, an endogenous concentration ofH₂O₂ is highly variable and can generally be lost in the processing ofthe plant material. One of the advantageous aspects of some embodimentsis that the source of the oxidizing agent can come from exogenouslyadded hydrogen peroxide, ozone, peroxone or other commercial oxidizer asthe activation precursor.

In some embodiments, a phenol unit can carry 1 to 3 hydroxyl groups (OH)that can react with the oxidizer. Therefore, a phenolic groups containedmacromolecule composed substantially of can carry hundreds to thousandsor more hydroxy groups. This high density molecular cluster of hydroxygroups provides various other non-covalent-bond opportunities, chargeattraction, and physical sequestration for hydrogen peroxide moleculeswithin close and reactive proximity to the phenolic units. Sequestrationin accordance with embodiments of the present invention provides a novelmethod of maintaining stability and enhanced saturation of hydrogenperoxide within the reactive proximity of the polyphenolic substrate atlower concentration solutions than the equivalent non-sequesteredcomponents. Sequestration can be the result of combining a sufficientlyhigh concentration of hydrogen peroxide in water to establishintermolecular attractive forces between a substantial portion of thepolyphenol hydroxyl functional groups and the hydrogen peroxidemolecules. Increasing sequestration can significantly reduce oxidizerloss from heating, ultraviolet exposure, reducing contaminants, andspontaneous degradation while increasing the potential number of highaffinity binding sites on the polymer upon encountering the appropriateenzymes.

In some embodiments, some amount of crude extracts of plant material,including the fragments of plant cells populated with partiallydenatured reactive oxygen species (ROS) processing enzymes likecatalase, peroxidase, dismutase, glucose oxidase, or a combinationthereof, can function as a hydrogen peroxide sequestration structure.These ROS include hydrogen peroxide (H₂O₂), superoxide (O₂.), singletoxygen (¹O₂*), and hydroxyl radical (.OH). In alternative embodiments,the activation precursor can be generated by the degradation ofdissolved ozone (O₃) or by its conversion by active dismutases intoH₂O₂. In other embodiments, the activation precursor can also begenerated by the enzymatic conversion of any superoxide, such as a fattyacid peroxide.

The component of the composition that contributes the phenol units cancontain a polyphenol and/or any heterogeneous or homogenousmacromolecule that is synthetic, plant, or animal derived. In someembodiments, the polyphenol contains more than two phenol groups. Inalternative embodiments, the polyphenol contains more than 30 phenolgroups. In other embodiments, the polyphenol contains from 100 to 10,000phenol groups. A person of ordinary skill in the art would appreciatethat any number of phenol groups can be contained in the composition solong as the compositions contain the desired features described herein,such as the rate of absorption is moderate.

Plant based materials described herein include, but not limited to,polyphenols, lignins, polysaccharides and other large molecule materialsor structures that are predominantly terminated by carbonyl groups thatare available for quinone transformation. Effective solutions can beprocessed from a vast variety of different plant tissues of differentspecies due the ubiquitous nature of appropriate materials.

The typical art for obtaining antimicrobial compounds from botanicalsources relies on mechanical or solvent extraction of organic moleculesin mariners that do not provide practical means for harnessing the woundresponse chemistry of living plant tissues for commercial application,especially in animal or other plant systems. Cellular disruption,whether from commercial processing, pathogen attack, herbivores,environmental damage or natural decomposition, triggers the reactionsand makes the antimicrobial compounds useless in a very short period oftime.

Polyphenols constitute a vast range of organic polymers produced byplants and are important substrates in the wound response of plants.These polymers are able to undergo some oxidative conversion to formantimicrobial compounds, but may produce slightly different behaviors.Polysaccharides may also undergo some oxidative transformation, formingbioadhesives with antimicrobial and wound sealing potential and can beeffective quinone analogues.

The underlying processes are rooted in a complex series of chemicalreactions that may involve multiple direct and indirect enzymaticformations of highly reactive intermediates. oxidoreductases can mediatethis chemical cascade, typically in the presence of a source of reactiveoxygen species, with several possible modalities converting hydroxyl(OH) functional groups of aromatic polymers into carbonyl groups (═O)that form covalent bonds responsible for high strength biologicstructures and antimicrobial defenses. Carbonyl groups are functionalgroups contain a carbon atom double-bonded to an oxygen atom: C═O.Ketone groups contain carbonyl groups (C—C(═O)—C) that each of thecarbonyl groups bonds to two other carbon atoms with the carbon atom ofthe carbonyl. Some of the particular interested substrates includecompounds with multiple aromatic subunits forming organic molecules withfully conjugated cyclic dione structures with >C(═O) groups in anyarrangement of double bonds, including polycyclic and heterocyclicanalogues, that form covalent bonds to nucleophilic amino acids andproteins. The term “quinonic” used in this document includes anycompounds containing subunits with any number of carbonyl groups.

The converted or oxidized hydroxyl subunits exhibit increased bindingaffinity characteristic of multiple quinones and semiquinones in aheteropolymer or homopolymer configuration. The oxidation is able toonly take place on a portion of the functional groups of a polyphenol.The typical heterogeneous character of these oxidized polyphenols isable to present quinonic activity while retaining some basic polyphenolbehavior. Differences in gross structure, tertiary form, and molecularweight will cause different affinities for different proteins withincreased gross mixture or intramolecular heterogenicity providingbroader spectrum potential.

Besides having been identified as a transient cofactor in a number ofbiologic cross-linking activities, quinone monomers have long been knownfor a very potent germicidal effect. In addition to provide a source ofstable free radicals, quinones are able to irreversibly complex withnucleophilic amino acids in proteins, often leading to inactivation ofthe protein and loss of associated biologic function. For that reason,the potential antimicrobial effect of quinonic compounds is great.Probable targets in the microbial cell are surface-exposed adhesions,cell wall polypeptides, motility effectors and membrane-bound enzymes.Quinones can also render substrates unavailable to the microorganism.

As with all plant-derived antimicrobials, the possible toxic effects ofquinones must be thoroughly examined. Some quinones have demonstratedantimicrobial effectiveness at 5 to 6 log dilution. Quinone monomers aresmall molecules that easily penetrate tissues and exhibit toxicity thatcan limit their medicinal use, whereas polyquinonic compounds contain amultitude of quinonic segments in a biopolymer, compound molecule, orsynthetic analog. Polyquinonic compounds of sufficient molecular weightand size can have reduced systemic absorption, corresponding to reducedtoxicity potential to higher organisms.

The study of germicidal activity of quinone compounds started as earlyas early 1900s but was not well understood until the 1940s. Formalininhibition of the color reaction between quinone and many differentproteins, such as egg albumin, casein, horse serum, and peptone,indicates that the reaction is principally between quinones and theamino group of proteins. The germicidal mechanism of a polyquinone alonecan take three primary forms: the covalent binding reaction withbacterial proteins, cross-linking of ruptured cell cyto-proteins toastringently form a barrier refractory to pathogens, and a REDOX cyclingmode that generates peroxides and free radicals that cause oxidativedamage to the pathogen envelope.

The major product of phenol oxidation was identified by Pryor in 1940 aso-diphenols. This production can result from auto-oxidation in thepresence of oxygen radicals or from enzyme conversion by phenoloxidases.Phenoloxidases (e.g., L-dopa: oxygen oxidoreductase; EC 1.14.18.1), alsoknown as polyphenol oxidases and tyrosinases (e.g., lysyl oxidase; EC1.4.3.13), are copper-containing proteins that catalyze the oxidation ofmonophenols into o-diphenols and the subsequent oxidation of o-diphenolsto the corresponding o-quinones. Phenoloxidases are widespread in theanimal kingdom, as well as in plants, fungi, and prokaryotes. Insectsalso use them in sclerotization in rapid formation of high strength eggcasings, cocoons and silk structures.

Even though they are the result of hundreds of millions of years ofdivergent evolution, the very close structural similarity of oxidativeenzymes including peroxidase, polyphenol oxidase, laccase, etc., foundin plant, fungi, bacteria and peroxidases such as myeloperoxidase,lactoperoxidase and all the peroxidases from different animal tissues,indicates the need to achieve the same basic biologic ends. Thisfunctional similarity can be utilized as a novel method for triggeringcontrolled oxidation of polyphenols across biologic kingdoms or phyla.

For example, tyrosinase is the core enzyme of the phenoloxidase familyalong with several other oxidoreductases that catalyze a step in theformation of melanin pigments. The tyrosinase in mammals is functionallysimilar to phenoloxidase in the chemical cascade that causessclerotization, melanization, and production of antimicrobial peptidesin insects.

Hydrogen peroxide is one of the most common biologic sources of reactiveoxygen species involved in the enzymatic creation of polyquinones. It isexpressed in substantial quantity in live tissues of many plants. It isa ubiquitous metabolic product and a key initiator that is consumed inthe polyphenol oxidation process that occurs in damaged tissues. Boththe oxidizer (H₂O₂) itself and its enzyme mediated reaction product,polyquinones, are antimicrobial with the latter also having strongastringent properties. These are the primary compounds that enable thetraditional medicinal use of many fresh plant materials as wounddressings. However, antimicrobial potential within the vicinity of afresh cut plant tissue is largely degraded within minutes due to thetransient nature of these compounds in wounded plant tissue.

The mixture of plant materials, oxidizers and enzymes have been used togenerate oxidized polyphenols and carbohydrates for a variety ofindustrial and commercial uses, but the composition of plant materialwith enzymes denatured or removed to allow stable combination withoxidizer for the purpose of applying to a target that provides aseparate source of catalyst or enzyme to affect biologic systems isnovel.

References describing compositions of plant materials and oxidizers foruse on biologic systems is limited. US 2002/0034553 describes an aloevera gel and Irish moss as a thickened passive carrier for deliveringhydrogen or zinc peroxide as a source of oxygenation to createunfavorable conditions for anaerobic bacteria on dermal wounds. U.S.Pat. No. 5,260,021 discloses a hydrogen peroxide-containing gel ointmentas a vehicle for carrying oxygen for use only as a disinfectant forcontact lenses or the like. U.S. Pat. No. 4,696,757 describes a hydrogenperoxide carrying gel for treating surface cuts and for bleaching hair.None of these patents make reference to combining an oxidizer, such ashydrogen peroxide, and a polyphenol component with the intent of causingor enabling a reaction between the two.

In higher plant physiology, hydrogen peroxide, polyphenols, proteins andoxidoreductases are segregated in the structured cytoplasm, organellesand membrane structures of the living cell. Disruption of the cell byinfection, injury, crushing, pulverizing, desiccating, ensiling or otherphysically damaging processes result in the mixing and exhausting of theuseful reactive potential of these components. The current art ofbotanical extraction offers no obvious means to capture a stablecombination of these components. It is therefore not surprising thatdespite over 50 years since the discovery and documentation of thefunction of oxidized polyphenol systems within plants, the botanicalhealth and agriculture industries have not successfully commercializedthis multi-molecular chemistry, instead focusing on capture ofinherently stable nutritional and pharmacologic molecules that can besimply packaged or extracted.

There is significant understanding of enzymatically oxidized polyphenolswithin the botanical sciences, but there is no known reference for astable ex-vivo method and composition for restoring, duplicating, orenhancing the capability of this polyphenol oxidation system for usewith animal physiology or other biologic applications outside thecontext of in-vivo plant biochemistry.

The environmental interfaces and immunologic needs of the plant kingdomare in many ways similar to those of animals. External plant tissues,such as leaf cuticles, fruit rinds, and seed husks, are living tissuesadapted to defend against similar pathogens and physical stresses ashuman derma and mucosa. Animals and plants also have some analogousmechanisms for coping with wounding. As such, the biochemical mechanismsused in the plants are able to be applied to animals.

In another aspect, a method of producing stable biochemical systemscomprises extracting a stable polyphenol substrate in a composition freeof active reducing agents, enzymes or catalysts and combining thepolyphenol substrate with a concentrated source of reactive oxygenspecies that promote initiation and propagation of oxidation reactionswhen applied to or combined with a separate source of appropriateoxidoreductases or catalysts.

Compositions in accordance with some embodiments of the inventionintroduce hydrogen peroxide in sufficiently high concentrations into anaqueous plant biopolymer solution to establish sequestering orstabilizing of concentrated oxidizers within intimate reactive proximityof the hydroxy functional groups of the polyphenols. The sequesteredoxidizer resists diffusion away from the biopolymer when subjected todilution. Reducing agents are removed or denatured in formulationscontaining the oxidizer-polyphenol combination to render theoxidizer-polyphenol combination substantially unreactive to proteinsuntil brought in contact with a surface, tissue, organism, coating orsolution that provides a source of oxidoreductase enzymes or othercatalytic agents that directly or indirectly mediate conversion of thebiopolymer into an activated form.

In another aspect, the compositions contain an efficient target-specificformation of oxidized biopolymers mediated by enzymes of animal origin.This mechanism enables the delivery of medically and commercially usefuleffects to animal body tracts or tissues. These effects are analogous tobasic plant wound responses. Many of the biochemical reactions involvedare well studied but still not fully understood. Nonetheless, there isno prior art for stabilizing and applying the stabilized combination ofoxidizers and polyphenols to animal physiology or pathogens as thesource of enzymatic activation. Common phenol complexing acting enzymesin many plants, bacteria, and fungi such as laccases or phenol oxidasesare functionally similar to common animal enzymes, such as catalases,peroxidases, that can also cause the formation of quinonic groups withenhanced affinity for amino acids of proteins.

In some embodiments, the liquid solution prepared has many significantpractical advantages over conventional typical antimicrobial botanicalextracts. These include increased water solubility and deliverability,broader pH stability, higher temperature stability, predictable potency,low, animal toxicity, love minimum inhibitory concentrations on a broadspectrum of gram negative and gram positive bacteria, low odor andtaste, efficiency in ease of raw materials, and lack of harmfulenvironmental breakdown products.

Some embodiments of the present invention mimic plant defense mechanismsthat have co-evolved with environmental pathogens over many millions ofyears. The non-specific protein binding action of the activatedbiopolymers contain multiple activities that can cause microbialinactivation. All are radically different than conventional germicidalor antibiotic actions and are far less likely to promote resistance in amanner that has been associated with subtherapeutic use of, andenvironmental contamination with, antibiotics.

The oxidized polyphenols can have several biologic mechanisms of action.A single such polyquinonic molecule can contain several to thousands ofbinding sites in a dense and non-diluting population, so that thecompound can cross-link and condense amino acids and proteins of damagedorganisms to form a refractory barrier against pathogens and irritants.The compound is able to bind to and cause significant distortion andinactivation of the metabolic pathways and virulence effects of thepathogens. They are able to physically agglomerate, entrain orimmobilize pathogens to prevent the pathogens from propagating. Quinonicor phenolic forms also can cause oxidative radical release to causepathogen membrane damage and lysis. They are also able to blockreceptors responsible for pain and inflammatory responses.

Polyphenols generated in accordance with embodiments of the inventioncan be individual soluble polymers, compound molecules, macromolecularconglomerates, or aggregates with proteins or cellular fragments.Stabilizing sequestration of the oxidizers can result from hydrogenaffinities, electron sharing, or from capillary effects on a largerscale.

In many situations, it can be beneficial to promote heterogeneousmixtures of activated biopolymers to maximizebio-activity/bioavailability against a broader range of pathogens. Thecombination of polyphenols extracted from multiple plant materials canserve to further broaden the binding spectrum. In some cases, not allphenol subunits undergo quinone conversion and the substrate can retaincharacteristics and behaviors of the unoxidized compounds in addition tothe quinonic behavior.

Those of ordinary skill in the art will understand that many othernatural or synthesized organic polymers can also be oxidized to form amultiplicity of carbonyl or quinonic functional groups that can be usedin place of or combined with polyphenols.

In some aspects, non-botanical enzymes are used to trigger the oxidationof polyphenols. This allows introduction of a stable polyphenol-oxidizersolution to passively traverse animal body tracts to deliver unreactedmaterials to compromised tissues that cannot be practically accessed byother means, thereby increasing the effective bioavailability of theuseful composition. Target catalyzed quinone formation constrains thenon-specific biochemical reactions to the immediate proximity ofcompromised tissue, thereby reducing the risk of undesirable effects ofgross tissue or systemic interaction.

The existence of functionally identical enzymes in the animal and plantkingdoms enable the novel transference of the enzyme mediated polyphenolreactions to non-botanical applications. For example, the G.I. tract ofhigher vertebrates has 2 different types of peroxidases. The first typecomprises a soluble peroxidase found in both rat and pig digestive mucusthat is secreted from the immune system eosinophil. The second typecomprises an insoluble peroxidase found in G. I. mucosal cells that areonly released in wounding. The second type of peroxidase is basicallythe functional equivalent to peroxidases involved in polyquinoneformation of plant wound response and can site-specifically triggerquinonic activity in the direct proximity of damaged, vulnerable, orinfected cells while remaining passive to healthy tissue.

Such a stable polyphenol-oxidizer composition has particularly usefulbiochemical effects on a tissue lesion or an irritated mucus membrane ofthe digestive tract, respiratory tract, urinary tract, reproductivetract, or other mucosal interfaces within higher animals. Ingestion ofthe composition in liquid solution will passively deliver it to the siteof damaged cells and infection where oxidoreductase enzymes willcatalyze polyquinonic activity. The polyquinones will cross-link andcondense cellular proteins of damaged host cells into a protectivebarrier. The non-specific high-affinity binding to surface proteins,enzymes, receptors, and structures critical to metabolism, virulence,and motility immobilize and inactivate a very broad spectrum ofpathogens. The agglomeration of pathogens can also increase thepotential antigenicity to evoke host immune responses.

In an aspect of the present invention, the strong but localizedastringent effect on the affected tissue can also reduce fluid exudates.Non-localized gross astringency from high concentrations of tannins andother polyphenols in the digestive system are known to interfere withnutritional absorptions and can cause mucosal damages; whereas,target-localized activation of quinonic behavior minimizes theseconcerns.

In some aspects, the compositions comprise novel bioactive microbialcontrol and tissue protective systems. The mixture of the bioactivematerial and activation precursors are catalyzed by various enzymes ofanimal, plant, or microbial cells. If the activating enzyme isassociated with tissue of interest such as wounded or infected tissue,the ingestion or application of the not activated composition will allowpassage to compromised tissue even deep in a body tract. Release ofoxidative radicals and formation of activated plant materialsite-specifically on target tissue significantly increasesbioavailability and constrains the enhanced activity to the immediateproximity of the enzyme source for reduced collateral interaction withother tissues.

In some of the embodiments, the bioactive plant-based material can bepolyphenols or any bio-molecules, synthetic polymers, aggregates ofsmall molecules, cellular fragments, or cross-linked groups of compoundscomprising a multiplicity to tens of thousands of exposed reacting sites

Many plants can be used as an inexpensive source of appropriatepolyphenols. Camilla Sinensis leaf is an example that can be a goodsource of a botanical raw material because of common availability ofcultivated sources, documented low natural toxicity and high watersoluble polyphenol content. The flavonol group of polyphenols(non-oxidized catechins) constitutes up to 30% of the dry leaf weight ofcamellia sinensis, making it an economical source. Effectiveantimicrobial astringent compositions have also been produced from manydifferent plant species and structures including rye seeds, mung beans,daikon skin, pomegranate rinds, bearberries, aloe vera skin, organ pipecactus, Chinese gall, oregano leaves, persimmon fruit, wheat germ,barley seeds, and coffee beans, demonstrating the ubiquitous nature ofthis plant defense mechanism in the plant kingdom.

In some aspects, methods of extracting a polyphenol substrate from plantmaterials enable stable formulation, storage, and delivery by havingextracts that contain substantially free active oxidoreductases or otherreducing agents. Some aspects of the invention involve thermal orsolvent denaturing of plant raw materials to obtain plant biopolymercomponent tree of active polyphenol oxidizing enzymes. Raw materialsupply availability and different plant tissue types can possiblydictate different processing. For instance, desiccated or dried plantmaterials are already devoid of hydrogen peroxide and therefore canundergo enzymatic denaturing processes after polyphenol extraction.

An example of an efficient process for producing an economicalpolyphenol raw material source free of degrading enzymes comprisesdesiccating freshly harvested whole camellia sinensis leaves rapidly inhigh temperature air to denature the polyphenol oxidases that can causeoxidation of green leaf polyphenols. This process maintains a very closecomposition to live tea leaves with the exception of the loss ofhydrogen peroxide, water, and a few enzymatic changes that typicallyoccur extremely rapidly upon harvest. The leaves are then pulverized tofacilitate handling and extraction efficiency.

In contrast, black tea undergoes an example of an alternativemanufacturing process. In the manufacturing process, Camilla sinensisleaves are crushed and their cellular structures are disrupted whilestill containing active polyphenol oxidase. This initiates enzymaticaerobic oxidation of catechins into quinones that spontaneously condenseto form volatile compounds. Plant material so processed is still able tobe a useful source of polyphenols, but the plant material will havelower content of polyphenols and will require additional enzymedenaturing by heating the plant material or its extract to a temperaturesufficient (preferably 80° C. to 110° C.) to blanch or denature theenzymes. A protein denaturing solvent, such as ethanol, is able to bealternately used in the plant material extraction process to destroy orremove cellular enzymes.

Most plants produce hydrogen peroxide as part of routine biologicactivities as well as in response to stress. The concentration of H₂O₂in plant tissue varies tremendously by species, tissue type,environmental stress and seasons. It is lost or consumed in typical postharvest processing and is generally impractical to capture from naturalsources, especially given the low cost of synthetic equivalentoxidizers.

In some embodiments, the methods and compositions in accordance withsome embodiments of the present invention comprise the exogenousaddition of hydrogen peroxide, which can be a commercially practical andstable source of reactive oxygen species for improved generation ofquinonic subunits within the polyphenols. A person of ordinary skill inthe art will also understand that other direct sources of reactiveoxygen species can be used for various applications, such as ozone, zincperoxide, peroxidases, carbamide peroxide, sodium percarbonate, calciumperoxide, magnesium peroxide, sodium perborate monohydrate, ozonide (O₃⁻), superoxide (O₂ ⁻), oxide (O²⁻), dioxygenyl (O₂ ⁺) or indirectsource(s) of reactive oxygen species, such as oxygen gas, disassociatedwater, catalytic decomposed fatty acids, glucose, and polyphenols areable to be used.

In some aspects, embodiments of the present invention includecombinations of high concentration reactive oxygen species, such ashydrogen peroxide, with a polyphenol substrate to provide a stabilizingenvironment that resists diffusion of hydrogen peroxide molecules awayfrom intimate reactive proximity to the phenol subunits. Polyphenolstructures provide many non-covalent bond opportunities and chargeattraction to hydrogen peroxide molecules. Hydrogen peroxide is also aproduct of the auto-oxidation of polyphenols, helping to maintain grossequilibrium in solution. Stable sequestrations are able to shield thehydrogen peroxide from heat, ultraviolet exposure, reducingcontaminants, and spontaneous degradation.

FIG. 1 illustrates several reaction pathways for phenol unit 102conversion into quinones 104 or semi-quinones 106 in accordance with oneembodiments of the invention. As used herein “quinone” refers to allquinonic compounds, such as quinones and semi-quinones. Hydrogenperoxide 108 can be both a source of reactive oxygen species forinitiating oxidation and can also be a product of polyphenol oxidation.Stable equilibrium in a solution with polyphenols can therefore beestablished. Once started, the source of reactive oxygen species (ROS)facilitates efficient propagation of quinone generation throughpolyphenol substrates even without direct enzymatic mediation.Concentrated H₂O₂ in water is therefore a good oxidizer component.However, hydrogen peroxide can be indirectly achieved through otherreactions, such as decomposition of ozone, fatty acids, or percarbonatesto name only a few such reactions. Cellular oxidoreductases can also beinvolved in the indirect generation of the oxygen species involved inthe initiation or propagation of the oxidation reaction cascade. Forexample, the catalase that defends animal cells and many pathogens fromROS damage will disassociate H₂O₂ into water and reactive oxygenspecies.

Hydrogen peroxide is naturally produced in plant and animal cells butits concentration can vary tremendously depending on species, season,stress, and tissue type. Although certain plant types, such assucculents, can store significant quantities of hydrogen peroxide intheir tissues, it is generally impractical to extract it from plantsources due to the presence of reducing enzymes segregated from thehydrogen peroxide and/or polyphenols only by delicate subcellulardivisions that are inevitably breathed by typical commercial extractionprocesses. Mixing triggers the oxidative wound response and rapidlyconsumes the hydrogen peroxide, leaving excess polyphenols, enzymes, andother non-involved botanical compounds.

Some aspects of the present application include the use of a separatelymanufactured or generated source of reactive oxygen species incombination with the polyphenol substrate substantially five of activereducing agents or enzymes.

In some embodiments, the oxidizer-biopolymer compositions are aqueoussolutions. In alternative embodiments, fibers, hydrogels, microporousmedia, micelles, emulsions, and other structures physically encapsulatethe biopolymer-oxidizer composition. In still other embodiments,mixtures of dry powders, granules, or other non-liquid polyphenolbearing materials combined with a dry oxidizer, such as potassiumpercarbonate, are used as a kit to be hydrated to produce a usefulpolyphenol-oxidizer solution.

In some embodiments, the catalyst is delivered to the target siteseparately as a liquid, aerosol, or as a surface coating on anapplicator, dressing, or cleaning implement. An example of this is anabsorbent sponge infused with a reducing agent, such as catalase orcopper, that will cause rapid release of oxygen radicals and quinoneformation when brought in contact with a polyphenol-oxidizercomposition. This can be used to generate a strong germicidal action,particularly to destroy viruses on non-biologic surfaces of to sanitizehealthy tissues in the absence of exposed catalyzing enzymes. Forexample, viral envelopes generally do not have enzymes, but are made upof proteins that can be bound by polyquinones. Thus, a separatelydelivered catalyst or enzyme can be used to initiate theviral/germicidal reactions.

In some aspects, polyquinonic compounds in accordance with theembodiments comprise utilities such as a microbial flocculant. Addingthese polyquinonic compounds to a contaminated water source can causeaggregation of the microorganisms into masses that either precipitateout or can be more easily filtered out by mechanical means. Thedeposition of polyquinonic compounds on mechanical filter media can trapproteins and microorganisms while imparting germicidal characteristics.This can be accomplished by applying an activated polyquinone to afilter media or circulating polyquinone forming compositions through afilter media that has a catalytic aspect to its surface such as abacterial biofilm. This can find application in many recirculating andsingle-pass filtration applications.

FIG. 2 illustrates a composition 200 prepared in accordance with someembodiments of the present invention. The composition 200 containsbiopolymers 202 containing hydroxyl group contained molecules 204. Thehydroxyl group contained molecules 204 are illustrated as polyphenols,but can be phenols, polyphenols, polysaccharides, or combinationsthereof. In alternative embodiments, the hydroxyl group containingmolecules 204 are tannins, lignins, and flavonoids. A person of ordinaryskill in the art would appreciate that the biopolymers 202 are able tobe any short linkage molecules (such as 2 to 100 repeating units or 100to1000 repeating units), macromolecules, long chain molecules, ringstructured molecules, π electron stacking and/or structural stackingmolecules. Further, the biopolymers 202 are also able to be anysubstance that can be derived or obtained from plants or artificialsynthetic molecules. Moreover, the biopolymers 202 are able to beobtained from combinations of plants. For example, the extract of plantA contains a high ratio of polyphenols and the extract of plant Bcontains a high ratio of polysaccharides. The biopolymer 202 can beobtained from a mixture of the extracts of both plant A and plant B. Insuch a case, 70% of the biopolymer 202 can come from plant A and 30% ofthe biopolymer 202 can come from plant B, so that the composition 200can have chemical properties closer to polyphenol than to the chemicalproperties of polysaccharides. Thus, desired reactive properties of thecomposition 200, such as a desired reactivity and reaction rate, can bedesigned by using different combinations of the plants A and B.

The composition 200 is also able to contain an oxidizing reagent 206and/or an enzyme 208. In some embodiments, the oxidizing agent 206comprises reactive oxygen species. In some embodiments, the oxidizingagent 206 comes from commercially available hydrogen peroxide 210, suchas >60%, 20%-60%, 35%, and 8%-20% of H₂O₂ in water. In alternativeembodiments, the oxidizing agent 206 comprises 1-2% or less than 10%H₂O₂ in water. In some embodiments, the oxidizing agent 206 comes from areaction of ozone 212, fatty acid 214, or percarbonate 216. In someembodiments, the oxidizing agent 206, such as hydrogen peroxide., isendogenously produced by the biopolymer or the plants. In alternativeembodiments, the oxidizing agent 206 is exogenously added to the system,such as by adding commercially available hydrogen peroxide to a solutionof the biopolymer 202 and the enzyme 208.

In some embodiments, the enzyme 208 is endogenously generated orexogenously added. For example, the enzyme 208 used to activate orfacilitate the reaction is generated by pathogens 230 on the tissue ofan animal 218. Alternatively, the enzyme 218 is generated at thecells/tissues of animals 218 and/or plants 232. Moreover, the enzyme canbe added to a solution containing the biopolymer 202 and the oxidizingagent 206 before applying the composition 200 to an animal or a plant.

In some embodiments, the hydroxy group contained molecule 204 formsquinonic compounds 234 and/or 246. The quinonic compound 234, 242, 244,246, hydroxy group 236, 238, 240, and the hydroxyl group containedmolecule 204 can provide interactions, such as covalent bonding forces,hydrogen bond interactions, or electron stacking interactions, to keepthe reactive oxygen species (ROS) localized in the reactive proximity.The reactive oxygen species can be oxidizing reagents.

FIG. 3 shows the step 300 of a process for preparing a plant-basedcomposition in accordance with one embodiment of the present invention.At Step 302, active reducing agents or catalysts are inactivated orremoved. At Step 304, polyphenol substrates substantially free of activereducing agents or catalysts are extracted. At Step 306, the polyphenolsubstrates are mixed with a source of reactive oxygen species and/orcatalysts for initiating and propagating of oxidation reactions. Asdescribed above, the source of reactive oxygen species, such as hydrogenperoxide, can be exogenously added or endogenously generated by a plantextract. Similarly, the catalysts for initiating the oxidation reactionscan be exogenously added or endogenously generated at an applied site,such as a pathogen-infected site or a tissue wounded area. At Step 308,the mixture is applied to a target site, such as a wounded area of ananimal. The method 300 described above is one embodiment. All the stepsare optional, and additional steps are able to be added. The sequence ofthe steps can be in any order. Other variations are applicable. Forexample, a solution, substantially free of reducing catalysts andcontaining polyphenols extracted from a plant, is mixed with hydrogenperoxide. The solution is able to be stored in a container for a lateractivation process. The solution is activated through an activationmechanism after being delivered to a pathogen-infected site of ananimal, which generate catalysts for activating the reactions. In otherexamples, a solution containing polyphenol and hydrogen peroxide isactivated by exogenous addition of catalysts before the application to atarget, such as a site of an animal or a plant.

FIG. 4 shows the step 400 of a process for inactivating reducingagents/enzymes by solvent in accordance with an embodiment of theinvention. At Step 402, the plants that are used to make the plant-basedcomposition are chosen. At Step 404, polyphenol compositions areextracted from the plant using a solution. At Step 406, the solution isheated to between 80° C. to 110° C. to blanch for denaturing theenzymes. At Step 408, a protein denaturing solvent, such as ethanol, isused to destroy or remove cellular enzymes contained in the plant.

FIG. 5 shows the step 500 of a process for inactivating reducingagents/enzymes by heat in accordance with an embodiment of theinvention. At Step 502, the plant used to make the plant-basedcomposition is chosen. At Step 504, the polyphenol oxidase from theplant is denatured by desiccating whole camellia sinensis leaves rapidlyin high temperature air. At Step 506, the water cellular enzyme isremoved and/or inactivated. At Step 508, the leaves are pulverized. AtStep 510, solvent is added to extract the polyphenols.

FIG. 6 illustrates some applications of the plant-based composition inaccordance with some embodiments. The applications include animalnutritional supplements, animal tissue coagulation, pathogen barrierformation, target triggering reactions, exogenous addition of oxidativeenzyme for biocidal applications, hydrogen peroxide sequestration, timeddelivery, pathogen metabolic halt, medicinal beverages, receptorantagonists, microbial flocculants, biological substance preservation,antimicrobial wash, agriculture applications, pond water sanitation,medical device surface treatments, and medicine delivery vehicles, toname only a few such applications.

Some modes of action in accordance with embodiments include the functionof target activation of a dense population of high-affinity,low-specificity binding sites on a relatively large bio-moleculesubstrate. In some embodiments, a whole plant extract is used. In someembodiments, a mixture of plants is blended with different dominantphenolic species and molecular weights with slightly different proteinaffinities, so the broadest possible range of activity can befacilitated.

Typically, the concept of drug design of conventional drugs has beenfocused on highly specific molecular interactions. In comparison, someembodiments of the invention use non-selective activity that is madehighly effective and safe by site-specific activation. In someembodiments of the present invention, the molecular complexes traversethe digestive system in a waterborne solution of molecules that keep thenecessary multiple reactive components sequestered in direct reactiveproximity of each other despite diffusion gradients in high levels ofdilution. This maintains full bio-availability until it encountersdamaged mucosal tissues or pathogens that present appropriate enzymes toactivate the complex. Activation at the site catalyzes a highlylocalized transformation of the passive molecular complex into anaggressive protein binder with hundreds or thousands of potential activesites, which are far more aggressive than the two binding sites found onantibodies. This highly site specific activation and non-absorbed natureof the large “sticky” polymer creates a powerful, precisely targetedaction that presents minimal adverse systemic potential. An accumulationof these “sticky” plant biopolymers becomes firmly anchored to thetarget site and starts to mimic the highly efficient mechanical immunereaction that would normally occur at the site of a plant injury.

In some embodiments, the mechanisms in plant immunology described aboveare applied to animals. The mechanisms include (1) non-specific bindingto functional pathways of infecting bacteria or yeasts, which can killthe bacteria or yeasts by impairing their metabolism and reproduction,(2) binding to toxins (generally proteinaceous) present at the siteand/or blocking pathogens from expression of more enzymatic virulencefactors, (3) immobilizing and/or impairing their motility or causingagglomeration that prevents propagation and shedding, (4) cross-linkingeffects of proteins of damaged cells into a physical barrier thatreduces exposure to further infection or irritation, (5) binding toinflammatory signal receptors functioning as antagonists for a varietyof physiologic responses, (6) disabling the cell penetration mechanismor entrapping viruses and/or preventing propagation and shedding, and(7) localized astringent and barrier effect to reduce interstitial fluidloss from damaged tissues.

In some embodiments, the effective dosages are extremely small comparedto physical astringents used in the traditional method to arrestdiarrhea and believed to be insufficient to create any physicalastringent effect that has been associated with intestinal damage ornutritional uptake impairment associated with the use of tannins.

Some experiments are performed using the complex prepared in accordancewith the embodiments of the present invention. The effectiveness of thecomplexes is supported by consistent observation of improved growth inpigs treated with the complexes made in accordance with some of theembodiments. The insensitivity of the complexes to dilution, the passivenature on healthy mucosa, and the minimal activity on non-pathogenicbacteria are some of the factors that enable and make low concentrationwaterborne delivery preferable and more effective.

The mixture prepared in accordance with some embodiments of the presentinvention can be useful on a tissue lesion or an irritated mucusmembrane of the digestive tract, respiratory tract, urinary tract,reproductive tract or other mucosal interface within higher animals.These tissues and infecting bacteria can be a source of oxidativeenzymes that catalyze the conversion of the polyphenol substrate intoits oxidized form. In some embodiments, the ingestion of thephenolic/oxidizer composition described above can direct and/orindirectly convert the polyphenols into a polyquinonic (or o-polyphenol)compound through direct enzymatic conversion or auto-oxidation ofhydroxyl units with an enzyme decomposed hydrogen peroxide. Thepolyquinonic molecule can then covalently bind to proteins of thedamaged tissue cells or the surface proteins of pathogens to immobilize,inactivate, and/or condense them. The bound proteins can form aprotective matrix, which reduces the ability of pathogens to colonize,provides a strong astringent effect that contracts the wound and reducesthe fluid exudate. The underlying reactions can take different pathways,depending on the specific enzymes, the structure and type of thepolyphenol molecule, the relative concentration of the oxidizer, thedilution environment, etc. An oxidizer that is not directly involved inenzymatic formation of quinonic units can release oxidative radicalsthat directly damage cellular structures of the pathogens. The localizedburst of such radicals in proximity of immobilized pathogens creates afocused germicidal action that is far more efficient than the diffuseaction of germicides in free solution. The use of more than one plantmaterial as substrates or combinations with plants with known germicidalactivity without oxidation as additional constituents can be desirablefor increasing the spectrum or potency of antimicrobial activity.

In other embodiments, the oxidative enzymes can be delivered to thereaction site separately. As an example, one of the embodiments containsfighting viruses in the absence of infected tissue or bacteria byproviding a source of catalyzing enzymes. Viral envelopes generally donot have the enzymes, but are made up of proteins that can be hound bythe macromolecule once converted into a polyquinone. The use of such apre-converted polyquinone is applicable in some embodiments.

Some embodiments of the present invention can be used in solution as agermicidal additive. It can be triggered by the enzymes ofmicroorganisms, such as bacteria and fungi, in the absence of otherplant or animal tissue. The germicidal effectiveness on surfaces or insolution can be further enhanced by the addition of an enzyme or othercatalyst to the surface or solution to be treated or by the applicationof the compositions to a surface, implement, or vessel that is treatedwith an activating enzyme or catalyst.

Some embodiments of the present invention can be used as a microbialfloculant, aggregating the microorganisms into masses that eitherprecipitate or can be filtered out by mechanical means. The aggregationof germs and their products can also increase the potential antigenicityto evoke immune responses. Astringent activity can be cytotoxic to thegerms as the result of (1) cross linking that disables the germ cells'surface proteins and enzymes or receptors, signal transductioncomponents, nutritional absorption and transfer functions and (2)reducing or impairing the mobility of germs and viruses. Both types ofactions can disable the infectivity of viruses.

The bactericidal test demonstrated that an amount of hydrogen peroxidethat can be exhausted by the bacteria in 16 hours can be used tocompletely kill all bacteria, without being consumed, when a compositionmade in accordance with the embodiments of the present invention isused. The plant-based material that did not undergo oxidizationreactions did not show any bactericide effect. This indicated that atleast some of the soluble polyphenolic compound was converted topolyquinone. In an in-vivo environment, this plant based germicidaleffect will be further increased if the amount of catalase or peroxidaseis not so limited. A transformed tobacco plant with a chimeric tobaccoanionic peroxidase is capable of producing a high level peroxidase,demonstrated to be 7 times faster at wound healing than anon-transferred tobacco plant. The increased efficiency of thepolyphenol to quinone conversion is more likely due to the significantamount of enzymes than to the amount of the substrate.

Some aspects of the present invention include the protein cross linkingcapability. Tannin has well known protein binding ability. However,tannin-protein binding is hydrogen bond based and the preferred bindingprotein is proline-rich-protein, usually in the saliva, and mucus fluidof digestive system. The protein cross linking and precipitation testdemonstrates that the egg albumin formed dense and completeprecipitation within 10 minutes. The precipitation with plants onlytakes 3 days to receive similar results. The sample with the oxidizingagent only no precipitation effect at all. This indicates theembodiments of the present invention have greatly increased proteincross linking capacity and efficiency.

Some of the embodiments include a mixture of a plant based germicidaland a protein cross linker composition and oxidizer. The plant basedgermicidal and protein cross linker composition can contain a planthaving high phenolic compounds. The oxidizer can be hydrogen peroxide.Some of the methods that can be used to manufacture such compositionsare disclosed in the U.S. patent application Ser. No. 12/317,638, filedDec. 23, 2008, and entitled “PLANT-BASED BIOCIDAL MATERIALS ANDSYSTEMS,” which is hereby incorporated by reference in its entirety.

Some embodiments of the present invention can be applied to theoxidation-reduction and/or radical reactions of the plant phenoliccompounds. The plant phenolic compounds can include phenols,polyphenols, hydroxycoumarin, flavonoids, alkaloids, and any othercomponents with chemical subunits similar to monophenols or diphenols tooxidize, to reduce, or to crosslink other molecules in or onmicroorganisms, plants, and animals, thereby, generating antimicrobialeffects, antiviral effects, astringent effects, and wound healingeffects on different target organisms or tissues. The terms “quinone”and “polyquinone” used in the present disclosure include any monomer ororganic polymer of aromatic ring configurations with one or more, doublebonded oxygen.

The compositions and methods can be utilized to facilitate the healingof the damaged wound on the skin or apply to the digestive system forhealing of any infection of the digestive system. The compositions andmethods can also be utilized in various targeted environmentalapplications, such as in-vitro and in-vivo uses.

In some embodiments, the applications of the present invention includeantimicrobials, anthelmintics, anti-laxatives/anti-diarrheas,analgesics, anti-inflammatories, cosmetic ingredients, keratolytics,oxidizers for industrial processes, metal chelating agents, and organicadhesives. In alternative embodiments, the physiologic uses includeantiseptics, disinfectants, virucides, fungicides, astringents, tissueadhesives, wound protectants, biofilm preventions, anti-inflammatories,analgesics, haemostasis, product preservatives, coagulants, flocculants,oral rinses, irrigants, debriding agents, gastric tonics,anti-diarrheal, ulcer treatments, sclerotizing agents, water sanitizers,water preservatives, oxidizing cleaners, and deodorizers. In alternativeembodiments, the applications include having the humans or animalsingest the compositions to treat or prevent pathogens or pathogenicmolecules from infecting, damaging, or being absorbed by the tissues oftheir digestive systems. In other embodiments, the applications includethe prevention or treatment of animal diarrhea through the reduction offluid secretions through astringent, anti-inflammatory, oranti-microbial action. In some embodiments, the applications include thetreatments of gastric reflux erosions, peptic ulcers, or other lesionsof digestive system. In alternative embodiments, the applicationsinclude the treatments of nasal or aural cavity irritations orinfections. In other embodiments, the applications include antimicrobialsprays to the respiratory tract to reduce the pathogens and also toprotect the respiratory tract lining from invasion by the pathogens,such as bacteria, viruses, and fungi. In some embodiments, theapplications include respiratory tract sprays and sinus rinses to flushthe contact allergens. In alternative embodiments, the applicationsinclude urinary tract rinses for anti-infective or anti-inflammatorytreatments or routine antiseptic rinses for urinary tract implant andkidney dialysis patients. In other embodiments, the applications includeantiseptic organ preservation for organ transplantation.

In some embodiments, the applications include an antimicrobial wash forbacteria, viruses, and yeast infections on normal or damaged skin,surface wounds, or in any mucosal cavity. In alternative embodiments,the applications include tissue adhesives for accelerated healing,closure, or haemostasis of surgical incisions or injuries. In otherembodiments, the applications include treatments of surgical incisionsor topical wounds for scar reduction. In some embodiments, theapplications include first aid treatment for topical cuts, burns, orabrasions. In alternative embodiments, the applications includeantiseptic salves, ointment rinses, or irrigants for oral mucosal ulcertreatment, and dental procedures. In other embodiments, the applicationsinclude periodontitis treatments and sensitive-tooth treatments, such astooth micro-crack sealing. In some embodiments, the applications includeoral rinses for halitosis. In alternative embodiments, the applicationsinclude soaks for dermatitis, jock itch, vaginal infections, andathlete's foot. In other embodiments, the applications include burn,chronic wound, and ulcer antimicrobial and healing treatments. In someembodiments, the applications include the prevention or reduction ofbiofilm formation on tissues or surfaces.

In alternative embodiments, the applications include agricultureapplications. For example, the surface pathogen can be reduced and themicro-wounds can be sealed by spraying or soaking the plant with thepolyphenol-oxidizer composition. Further, the general health of theplants can be improved by strengthening the surface structure orstimulating enhanced growth or development by the crosslinkingreactions.

In other embodiments, the applications include aerosol or liquid spraysof the composition as a bio-security sanitizer for animal farmfacilities. In some embodiments, the applications include animal feedsterilization. In alternative embodiments, the applications include foodor water additives for preservation and prevention of diseasetransmission. In other embodiments, the applications include plant,fresh fruit, and vegetable washes. The spraying or rinsing a solutioncontaining the composition disclosed herein can kill or suppress surfacebacteria, extend shelf life, and protect the surface from or deterpest-invasion in live crops or agricultural produce. In someembodiments, the applications include plant seed disinfection forstorage and sanitation before germination. In alternative embodiments,the applications include preservation spray or water treatment forfreshly cut flowers. In other embodiments, the applications includetissue adhesive for plant grafting and groundwater remediation.

In some embodiments, the applications include meat and sea foodpreservation spray to reduce bacteria and to form thin anti-digestivelayers to prevent a microbial invasion. The alternative embodimentsinclude meat processing sanitizers for prevention of microbialcontamination. In alternative embodiments, the applications include pondwater sanitation for fisheries, such as fish, shrimp, oyster, abalone,and mussels. In other embodiments, the applications include diseasetreatment for aquatic plants and animals. In some embodiments, theapplications include aquarium sanitizers, preservative additives forliquid-containing products, disinfectant ingredients for surfacecleaners, quinone REDOX cycling coatings for medical devices, clothingand food preparation equipment, hospital environment and instrumentsanitization, antimicrobial hydrating solutions for hydrophilic coatedmedical devices, and organic anti-corrosive treatments for metals.

In alternative embodiments, the applications include industrial watershock, preservatives, or antifoulants. In other embodiments, theapplications include hot rub and swimming pool water sanitation. In someembodiments, the applications include carriers for small moleculetherapeutic compounds. In alternative embodiments, the applicationsinclude stabilizers for oxidizers, modification of food flavors, andinjection into tumors and cysts.

In some embodiments, the compositions are able to be in a dry powderform. The composition is able to be fed to an animal in a dry powderform or in a combination with at least one fluid. The compositions in adried form are able to contain polyphenol or polymeric molecules,reactive oxygen species, catalysts, or a combination thereof. Thereactive oxygen species can contain sodium percarbonate, potassiumpercarbonate and/or any other substance that is capable of activatingthe polyphenol and/or the polymeric molecules. The reactive oxygenspecies, the material containing polyphenol or polymeric molecules, acatalyst, or a combination thereof are able to be fed to animalsconcurrently or separately.

In some embodiments, the term “polyphenols” used herein contains morethan one phenol unit or building block per molecule. In alternativeembodiments, the term “polyphenols” contains one phenol unit permolecule. In other embodiments, the term “polyphenols” includeshydrolysable tannins (Gallic acid esters of glucose and other sugars)and phenylpropanoids, such as lignins, flavonoids, and condensedtannins.

In some embodiments, the hydrogen peroxide added to a polyphenolcontained solution is 1-2%. In alternative embodiments, the hydrogenperoxide added to a polyphenol contained solution is less than 10%. Aperson who has ordinary skill in the art would appreciate that anyconcentrations of hydrogen peroxide are applicable so long as theconcentration of the hydrogen peroxide is not too high to overreact withthe polyphenols in the solution. The overreacting reactions includeproviding a concentration of hydrogen peroxide, which makes theactivated polyphenols unable to perform the functions described in thepresent application.

In some embodiments, the term “fluid” used herein includes liquid, gas,supercritical fluid, a mobile solid form of substance, or a combinationthereof. In some embodiments, the term “pathogen” includes anyinfectious agents, gems, bacteria, virus, or a combination thereof. Insome embodiments, the term “pathogen” includes any biological substancesthat can potentially cause disease, illness, damage, harm, or negativeimpact to a host such as an animal or another biological substance. Insome embodiments, the term “biopolymer” includes any substances that canbe derived or obtained from a plant, an animal, or biologicalsubstances. In alternative embodiments, the term “biopolymer” includesany polymeric molecules produced by a biological organism, such as alive plant. Further, the term “biopolymer” can include cellulose andstarch, proteins and peptides, and DNA and RNA. In some embodiments, theterm “biopolymer” includes plural units of sugars, amino acids, andnucleotides. In some embodiments, the term “binding affinity” includesany intermolecular or intramolecular interactions and/or bondings. Forexample, covalent bonds, ionic bonds, hydrogen bonds, dipole moments,induced dipole moments, and electrostatic forces. In some embodiments,the term “reactive proximity” refers to any interaction that existsbetween two or more molecules/atoms, so that the two or more moleculesare not randomly freely moving in a solution.

In some embodiments, the term “effect in inactivating a pathogen”disclosed herein includes blocking pathogens from accessing animal/planttissues, smothering the metabolic pathways of the pathogens, bindingviral factors, immobilizing/aggregating the pathogens, and/or performingoxidative damages to the pathogens. In some embodiments, the sources ofreactive oxygen species and/or hydrogen peroxide are able to be obtainedfrom natural and artificial sources, such as a flesh aloe and/orcilantro. In some embodiments, the activated polyphenol includeo-polyphenol, oxidized polyphenol, polyphenone, and polyquinone.

The term “process fluid” is able to include a fluid that is artificiallyand/or biologically processed. The term “biologically processed” is ableto refer that an added composition is processed by a biologicalsubstance, such as an animal. The term “artificially processed” is ableto include filtration, desiccation, isolation, extraction, or any othermanufacturing or chemical/biological lab processes. For example, theterm “process fluid” is able to include the situation that a dry powderform of substances is fed to animals and having a fluid of the animalsor added fluid to dissolve or disperse the dry powder, thereby forming aprocessed fluid. The dry powder is able to contain both polyphenols andreactive oxygen species, such as sodium percarbonate and potassiumpercarbonate, to be fed to the animals. Alternatively, the dry powder isable to contain mainly polyphenols. The reactive oxygen species is ableto be fed to the animal in a liquid form together or separately with thedry powder. In another alternative embodiment, the dry powder form isable to contain mainly reactive oxygen species. The polyphenols orhydroxyl groups contained molecules are able to be fed to the animals ina liquid form together or separately with the dry powder.

The following experiments show the effectiveness of the compositions prepared in accordance with some of the embodiments of the presentinvention.

Experiment 1

Three samples of purified powdered bovine serum albumin (BSA) wereprepared in aqueous solution. Sample #1 contained BSA only. Sample #2contained BSA and polyphenol oxidase. Sample #3 contained BSA,polyphenol oxidase, and hydrogen peroxide. Each sample showed similarsteady state turbidity after 30 minutes as measured by aspectrophotometer. An aqueous solution of polyphenols (tannin) fromChinese Gall was added to each of the samples. After one hour, Sample #1showed little visible change. Sample #2 exhibited an increase inturbidity from increased particle size, indicating minor proteincoagulation. Sample #3 exhibited heavy precipitate on the bottom of thetest tube and a lack of turbidity, demonstrating that a substantialincrease in protein coagulation can be achieved by the enzymaticreaction of polyphenols with a source of reactive oxygen species.

Experiment 2

A solution of Chinese Gall and hydrogen peroxide was added to (1) a tubecontaining powdered chicken egg white (the desiccation processes used inmanufacturing powdered egg white denatures enzymes) reconstituted inwater and (2) a tube containing fresh chicken egg albumin in water. Muchgreater precipitation was observed in the fresh chicken egg albuminsample, demonstrating that plant polyphenols can be catalyzed by enzymesof animal origin to increase protein binding consistent with quinoneformation exhibited in plant wounding.

Experiment 3

Formulation A was prepared using the method described below. One gram ofcommercial green tea powder was prepared in 1 liter of deionized waterin a 1 liter Pyrex beaker and allowed to extract at room temperature for6 hours. 35% food grade hydrogen peroxide was added to the solution andallowed to sit for 4 hours, then filtered through a 2 micron mesh mediaor filter. The resulting stock solution was diluted 1000:1, 200:1, and100:1 with 18 Mohm water. 15 ml of dilute solutions was added to equalpart culture solutions containing 10 e7 wild strain E. coli cultures(water controls) and allowed to incubate at 37° C. At 2 hr, 4 hr, 6 hr,and 8 hrs, a sample from each test series was flooded on agar plates andincubated, and manual colony counts were performed. The 100:1 sampleachieved 100% kill at 4 hrs, the 200:1 achieved 100% kill at 6 hrs, andthe 1000:1 was only bacteriostatic. This demonstrates the feasibility ofmanufacturing a botanical based composition with high germicidalcapabilities with exceptionally low raw material and energy input.

Experiment 4

A wild strain E. coli culture was added to three samples. The sample Acontained 25 ppm hydrogen peroxide in water. The sample B contained asolution of Formulation A diluted to equivalent 25 ppm hydrogen peroxidefraction. The sample C contained a solution having a green tea extractof the same concentration of Formulation A but without hydrogenperoxide. The bacteria's population in the hydrogen peroxide (the sampleA) was initially reduced but began to exhibit increased visual turbidityafter 16 hours, indicating the exhaustion of antimicrobial capability.The green tea extract alone (the sample C) exhibited little noticeableantimicrobial activity. Formulation A (the sample B) continued to killthe bacteria and showed no rebound after 3 days, indicating 100% killand/or increased antimicrobial effectiveness, which demonstratessignificantly enhanced germicidal performance resulting from thecombination of green tea extract and hydrogen peroxide.

Experiment 5

Sample #2 was a pre-prepared solution using the Formulation A above,which was diluted to duplicate the same polyphenol concentration as apre-prepared Sample #1. In Sample #3, polyphenol and dilute hydrogenperoxide were combined to achieve the same ratio of polyphenol tohydrogen peroxide as in Sample #2. Serial dilutions of each sample wereprepared and left standing for 24 hours. A solution containing 10e5/mlwild strain E. coli was added to each sample, which was then incubatedand plated out on agar for visual colony counts. The pre-preparedpolyphenol-oxidizer solutions of Sample #2 continued to kill bacteriaeffectively at significantly lower concentrations than in the Sample #3dilutions, demonstrating the enhanced performance at low concentrationswhen a polyphenol substrate-oxidizer composition is produced at a highconcentration (in the absence of active oxidoreductases or otherreducing agents) before dilution, supporting the concept ofintermolecular force sequestration for improved stability.

Experiment 6

The following is a method of preparing Formulation B and a demonstrationof a polyphenol substrate extraction process. 30 grams of driedpomegranate rind is used, which was dried at 150 C for 1 hr, grounded tofine powder, and combined with 10 liters of deionized water heated to 80C for 20 minutes, then was cooled to room temperature for 2 hours. 35%food grade hydrogen peroxide was added and allowed to sit for 6 hrs, andthen filtered through a 5 micron media. The added hydrogen peroxidesolution was less than 10% in solution after added into the solution toprevent overreacting with the polyphenols. Serial water dilutions of theresultant solution were added to 10e7-10e8 liquid bacteria cultures. Theresultant solution was incubated for 24 hrs and visually observed forturbidity. As shown in Table 1, the turbidity (+ turbid, − not turbid)indicates viable bacteria.

TABLE 1 Concentration (ug/ml) Bacterium species 500 250 125 62.5 31.315.6 7.81 3.9 1.95 0.98 0.49 Control Escherichia coli − − − − − − −− + + + + Salmonella enterica St. Typh. − − − − − − − + + + + +Staphylococcus aureus − − − − − − − − + + + + Pseudomonas aeruginosa − −− − − − − + + + + + Listeria monocytogenes − − − − − − + + + + + +Pasteurella multocida − − − − − − − + + + + + Proteus vulgaris − − − −− + + + + + + + Klebsiella pneumoniae − − − − − − − − + + + + Bacilluscercus − − − − − − − − + + + + Bordetella brochiseptica − − − − − − −− + + + +

Experiment 7

The application of a 200:1 dilution of formulation A to bilateralsymmetric lancet wounds on laboratory mice demonstrated wound closure inapproximately one third of the wounds treated with a saline control orantibiotic ointment.

Experiment 8

A cotton pad soaked in a 200:1 dilution of Formulation A was applied toinflamed oral mucosa for 10 minutes. Of three volunteers, allexperienced significant reduction in pain and swelling within one hour.Infection was completely resolved in two, demonstratinganti-inflammatory and anti-infective potential on mucosal tissue.

Experiment 9

Ten human volunteers with current and a history of past symptoms offrequent or chronic diarrhea were given 5 ml of 40:1 dilution ofFormulation B in 250 ml of water for 5 days. Nine expressed significantreduction in discomfort and symptoms for 1 week or more.

Experiment 10

0.5 ml of 100:1 dilution of Formulation A and Formulation B wereintroduced into a punch well on a sheep blood infused agar plate thatwas surface inoculated with E. coli. The perimeter of the well quicklyformed an opaque area of cross linked blood proteins that wererefractory to penetration of the PP-O complex and showed no bacterialsuppression zone around the well. In comparison, minimal nutrient agarwithout blood proteins showed a significant suppression zone indicativeof PP-O diffusion through the medium, demonstrating the low tissuepenetration of the compositions supporting reduced toxicity potential.

EXAMPLE 11

5-day-old Asian Landrace hybrid pigs in a commercial farm were dividedinto 13 test subjects and 3 control subjects, all fed identicalquantities and types of feed from birth to 3 months. Test subjects weregiven 5 micrograms (dry plant weight equivalent) of Formulation A everythird day in feed water and the same dose daily if diarrhea wasobserved. Controls were given antibiotic injections to treat diarrhea.At 21 days, the test group encountered less diarrhea and an average were1.0 kg heavier than the controls. At 3 months the average weight of thetest subjects was between 25-30% greater than that of the controls basedon girth measure, demonstrating Formulation A's viability as analternative to antibiotics as a prophylactic and growth promoter.

Experiment 12

The growth of 99 purebred Landrace piglets was tracked acid evaluated.The piglets were fed an oxidizer-polyphenol composition of Formulation Bin Swine milk replacer. 21-day-old starters tend to be stressed byenvironmental transition and have increased incidences of diarrhea forapproximately one week. The experimental group showed 18% higher averageweight gain during this period than the control group, demonstratingcommercial value in growth optimization and compatibility with milkreplacer.

TABLE 2 Experimental Group Control Group No. of Piglets 53 46 Dosage 7.5μg (dry plant wt) 0 μg Frequency Once per day None Testing period 8 days8 days Avg. wt. at beginning 7.00 kg 7.225 kg Avg. Wt. at the end 8.81kg 8.76 kg Average weight gain 1.81 kg 1.535 kg

Experiment 13

Toxicological safety was tested using 10 specific pathogen-free purebredlandrace pigs, each 23 days old. The pigs were administered one 250 μgor one 2500 μg, dose daily for 45 days. Hematology and growth weremonitored and histology performed, showing no negative effect on tissueor organs.

Experiment 14

50 weaned starter piglets were divided into 5 groups and each given 12μg dose once every third day for 5 weeks. Statistical analysis showssuperior weight gain in experimental groups. The above experiments areevidence of the effective uses of polyphenol-oxidizer compositions inthe control of pathogens and show measurable value in numerouscommercial and medically useful applications. Although the experimentsdemonstrate direct growth promotion benefits in agricultural animalproduction, the use of swine are known to have close physiologic andimmunologic similarities to humans and are commonly used as predictorsof performance and safety in humans. Potential effects can therefore beprojected and claimed for humans. This is supported by direct experiencein the rapid quelling of occasional digestive discomfort and diarrhea ofnon-specific causes. Single doses ranging from 20 μg to 250 μg (dry wt.equiv.) have been observed to be effective in resolving diarrhea inhumans, with symptomatic relief typically noticed within one hour.

Production

Some of the embodiments have been produced according to the methodsdescribed herein. The manufacturing process can use organic productionmaterials and procedures using National Organic Program (NOP) approvedand Generally recognized as Safe (GRAS) food-grade materials. Themanufacturing process can be carried out in a clean-room laboratoryunder Good Manufacturing Practice (GMP).

Some methods of production include botanical pre-process, proteindenaturization and extraction, intramolecular sequestration of reactantsinto meta-stable phenolic complex, post processing, and dilution andformulation with additional ingredients.

The process disclosed herein is applicable to a wide range of plantspecies and tissue types due to the ubiquitous nature of the chemistryof interest. The sources of the plant can be chosen based on theavailability of cultivation sources, key fraction content and secondaryconstituents that can potentially impart desirable or undesirablecharacteristic effects, such as toxicity and auto-degradation.

The manufacturing of the composition disclosed herein can incorporateseveral standardized quality control methods that directly measuresubstrate contents, initiator potency, microbiologic performance, andmolecular binding capacity. The manufacturing of the composition hadbeen tested in room temperature for over a year. The result showsaccelerated stability testing with retained samples. The microbiologicstability dilution threshold of the compound has been determined to beabove that which makes it is self preserving.

Some of the embodiments have been successfully produced from a varietydifferent plant bases. Preferred results are obtained whennon-controversial well characterized food plants with a long history ofuse in complementary and alternative medicines are used. A person ofordinary skill in the art would appreciate that many plants, planttissues or combinations can be used to make different formulations fordifferent markets, as long as the working mechanism is functionally,structurally, chemically, biologically, or physically equivalent to theembodiments described herein.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding ofprinciples of construction and operation of the invention. Suchreference herein to specific embodiments and details thereof is notintended to limit the scope of the claims appended hereto. It will bereadily apparent to one skilled in the art that various modificationsmay be made to the embodiments chosen for illustration without departingfrom the spirit and scope of the invention as defined by the appendedclaims.

We claim:
 1. A method of blocking a gram-negative bacteria fromexpressing a virulence factor, the method comprising contacting thegram-negative bacteria with an aqueous solution having a combination ofa water soluble polyphenol and hydrogen peroxide.
 2. The method of claim1, further comprising creating the aqueous solution from a dry, stableform of the composition.
 3. The method of claim 2, wherein the creatingincludes obtaining the dry, stable form of the composition as a drymixture of the polyphenol and hydrogen peroxide.
 4. The method of claim2, wherein the creating includes obtaining a dry, water solublepolyphenol; obtaining a dry source of hydrogen peroxide selected fromthe group consisting of sodium percarbonate, potassium percarbonate, acarbamide peroxide, and urea peroxide; and, combining the dry, watersoluble polyphenol and the dry source of hydrogen peroxide to create theaqueous solution.
 5. The method of claim 1, wherein the contactingincludes treating a digestive system of a subject for diarrhea.
 6. Themethod of claim 1, wherein the contacting includes treating an infectedtissue.
 7. The method of claim 1, wherein the contacting includesaccelerating wound healing.